The present invention relates generally to methods of treating microorganism-caused infections using cationic peptides or a combination of cationic peptides and antibiotic agents, and more particularly to using these peptides and antibiotic agents to overcome acquired resistance, tolerance, and inherent resistance of an infective organism to the antibiotic agent.
For most healthy individuals, infections are irritating, but not generally life-threatening. Many infections are successfully combated by the immune system of the individual. Treatment is an adjunct and is generally readily available in developed countries. However, infectious diseases are a serious concern in developing countries and in immunocompromised individuals.
In developing countries, the lack of adequate sanitation and consequent poor hygiene provide an environment that fosters bacterial, parasitic, fungal and viral infections. Poor hygiene and nutritional deficiencies may diminish the effectiveness of natural barriers, such as skin and mucous membranes, to invasion by infectious agents or the ability of the immune system to clear the agents. As well, a constant onslaught of pathogens may stress the immune system defenses of antibody production and phagocytic cells (e.g., polymorphic neutrophils) to subnormal levels. A breakdown of host defenses can also occur due to conditions such as circulatory disturbances, mechanical obstruction, fatigue, smoking, excessive drinking, genetic defects, AIDS, bone marrow transplant, cancer, and diabetes. An increasingly prevalent problem in the world is opportunistic infections in individuals who are HIV positive.
Although vaccines may be available to protect against some of these organisms, vaccinations are not always feasible, due to factors such as inadequate delivery mechanisms and economic poverty, or effective, due to factors such as delivery too late in the infection, inability of the patient to mount an immune response to the vaccine, or evolution of the pathogen. For other pathogenic agents, no vaccines are available. When protection against infection is not possible, treatment of infection is generally pursued. The major weapon in the arsenal of treatments is antibiotics. While antibiotics have proved effective against many bacteria and thus saved countless lives, they are not a panacea. The overuse of antibiotics in certain situations has promoted the spread of resistant bacterial strains. And of great importance, antibacterials are useless against viral infections.
A variety of organisms make cationic (positively charged) peptides, molecules used as part of a non-specific defense mechanism against microorganisms. When isolated, these peptides are toxic to a wide variety of microorganisms, including bacteria, fungi, and certain enveloped viruses. One cationic peptide found in neutrophils is indolicidin. While indolicidin acts against many pathogens, notable exceptions and varying degrees of toxicity exist.
Although cationic peptides show efficacy in vitro against a variety of pathogenic cells including gram-positive bacteria, gram-negative bacteria, and fungi, these peptides are generally toxic to mammals when injected, and therapeutic indices are usually quite small. Approaches to reducing toxicity have included development of a derivative or delivery system that masks structural elements involved in the toxic response or that improves the efficacy at lower doses. Other approaches under evaluation include liposomes and micellular systems to improve the clinical effects of peptides, proteins, and hydrophobic drugs, and cyclodextrins to sequester hydrophobic surfaces during administration in aqueous media. For example, attachment of polyethylene glycol (PEG) polymers, most often by modification of amino groups, improves the medicinal value of some proteins such as asparaginase and adenosine deaminase, and increases circulatory half-lives of peptides such as interleukins.
None of these approaches are shown to improve administration of cationic peptides. For example, methods for the stepwise synthesis of polysorbate derivatives that can modify peptides by acylation reactions have been developed, but acylation alters the charge of a modified cationic peptide and frequently reduces or eliminates the antimicrobial activity of the compound. Thus, for delivery of cationic peptides, as well as other peptides and proteins, there is a need for a system combining the properties of increased circulatory half-lives with the ability to form a micellular structure.
The present invention discloses analogues of indolicidin, designed to broaden its range and effectiveness, and further provide other related advantages. The present invention also provides methods and compositions for modifying peptides, proteins, antibiotics and the like to reduce toxicity, as well as providing other advantages.
In addition neither antibiotic therapy alone of cationic peptide therapy alone can effectively combat all infections. By expanding the categories of microorganisms that respond to therapy, or by overcoming the resistance of a microorganism to antibiotic agents, health and welfare will be improved. Additionally quality of life will be improved, due to, for example, decreased duration of therapy, reduced hospital stay including high-care facilities, with the concomitant reduced risk of serious nosocomial (hospital-acquired) infections.
The present invention discloses cationic peptides, including analogues of indolicidin, cecropin/melittin fusion peptides, in combination with antibiotics such that the combination either synergistic, able to overcome microorganismal tolerance, able to overcome resistance to antibiotic treatment, or further provides other related advantages.
The present invention generally provides the co-administration of cationic peptides with an antibiotic agent and also provides indolicidin analogues.
In related aspects, an indolicidin analogue is provided, comprising up to 25 amino acids and containing the formula: RXZXXZXB(SEQ ID NO: 1); BXZXXZXB(SEQ ID NO: 2) wherein at least one Z is valine; BBBXZXXZXB(SEQ ID NO: 3); BXZXXZXBBBn(AA)nMILBBAGS(SEQ ID NO: 5-8); BXZXXZXBB(AA)nM(SEQ ID NO: 9-10); LBBnXZnXXZnXRK(SEQ ID NO: 18-18); LKnXZXXZXRRK(SEQ ID NO: 19-20); BBXZXXZXBBB(SEQ ID NO: 21), wherein at least two X residues are phenylalanine; BBXZXXZXBBB(SEQ ID NO: 22), wherein at least two X residues are tyrosine; and wherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; AA is any amino acid, and n is 0 or 1. In preferred embodiments, Z is proline, X is tryptophan and B is arginine or lysine. In other aspects, indolicidin analogues having specific sequences are provided. In certain embodiments, the indolicidin analogues are coupled to form a branched peptide. In other embodiments, the analogue has one or more amino acids altered to a corresponding D-amino acid, and in certain preferred embodiments, the N-terminal and/or the C-terminal amino acid is a D-amino acid. Other preferred modifications include analogues that are acetylated at the N-terminal amino acid, amidated at the C-terminal amino acid, esterified at the C-terminal amino acid, modified by incorporation of homoserine/homoserine lactone at the C-terminal amino acid, and conjugated with polyethylene glycol or derivatives thereof.
In other aspects, the invention provides an isolated nucleic acid molecule whose sequence comprises one or more coding sequences of the indolicidin analogues, expression vectors, and host cells transfected or transformed with the expression vector.
Other aspects provide a pharmaceutical composition comprising at least one indolicidin analogue and a physiologically acceptable buffer, optionally comprising an antibiotic agent.
In other embodiments, the pharmaceutical composition further comprises an antiviral agent, an antiparasitic agent; and an antifungal agent. In yet other embodiments, the composition is incorporated in a liposome or a slow-release vehicle.
In yet another aspect, the invention provides a method of treating an infection, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition. The infection may be caused by, for example, a microorganism, such as a bacterium (e.g., Gram-negative or Gram-positive bacterium or anaerobe; parasite or virus.
In other aspects, a composition is provided, comprising an indolicidin analogue and an antibiotic. In addition, a device, which may be a medical device, is provided that is coated with the indolicidin analogue and may further comprise an antibiotic agent.
In other aspects, antibodies that react specifically with any one of the analogues described herein are provided. The antibody is preferably a monoclonal antibody or single chain antibody.
In a preferred aspect, the invention provides a composition comprising a compound modified by derivatization of an amino group with a conjugate comprising activated polyoxyalkylene and a lipophilic moiety. In preferred embodiments, the conjugate comprises sorbitan linking polyoxyalkylene glycol and fatty acid, and more preferably is polysorbate. In preferred embodiments, the fatty acid is from 12-18 carbons, and the polyoxyalkylene glycol is polyoxyethylene, such as with a chain length of from 2 to 100. In certain embodiments, the compound is a peptide or protein, such as a cationic peptide (e.g., indolicidin or an indolicidin analogue). In preferred embodiments, the polyoxyalkylene glycol is activated by irradiation with ultraviolet light or by treatment with ammonium persulfate.
The invention also provides a method of making a compound modified with a conjugate of an activated polyoxyalkylene and a lipophilic moiety, comprising: (a) freezing a mixture of the conjugate of an activated polyoxyalkylene and lipophilic moiety with the compound; and (b) lyophilizing the frozen mixture; wherein the compound has a free amino group. In preferred embodiments, the compound is a peptide or antibiotic. In other preferred embodiments, the mixture in step (a) is in an acetate buffer. In a related aspect, the method comprises mixing the conjugate of an activated polyoxyalkylene and lipophilic moiety with the compound; for a time sufficient to form modified compounds, wherein the mixture is in a carbonate buffer having a pH greater than 8.5 and the compound has a free amino group. The modified compound may be isolated by reversed-phase HPLC and/or precipitation from an organic solvent.
The invention also provides a pharmaceutical composition comprising at least one modified compound and a physiologically acceptable buffer, and in certain embodiments, further comprises an antibiotic agent, antiviral agent, an antiparasitic agent, and/or antifimgal agent. The composition may be used to treat an infection, such as those caused by a microorganism (e.g., bacterium, fungus, parasite and virus).
This invention also generally provides methods for treating infections caused by a microorganism using a combination of cationic peptides and antibiotic agents. In one aspect, the method comprises administering to a patient a therapeutically effective dose of a combination of an antibiotic agent and a cationic peptide, wherein administration of an antibiotic agent alone is ineffective. Preferred peptides are provided.
In another aspect, a method of enhancing the activity of an antibiotic agent against an infection in a patient caused by a microorganism is provided, comprising administering to the patient a therapeutically effective dose of the antibiotic agent and a cationic peptide. In yet another aspect, a method is provided for enhancing the antibiotic activity of lysozyme or nisin, comprising administering lysozyme or nisin with a cationic peptide.
In other aspects, methods of treating an infection in a patient caused by a bacteria that is tolerant to an antibiotic agent, caused by a microorganism that is inherently resistant to an antibiotic agent; or caused by a microorganism that has acquired resistance to an antibiotic agent; comprises administering to the patient a therapeutically effective dose of the antibiotic agent and a cationic peptide, thereby overcoming tolerance, inherent or acquired resistance to the antibiotic agent.
In yet other related aspects, methods are provided for killing a microorganism that is tolerant, inherently resistant, or has acquired resistance to an antibiotic agent, comprising contacting the microorganism with the antibiotic agent and a cationic peptide, thereby overcoming tolerance, inherent resistance or acquired resistance to the antibiotic agent.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.